Journal: The Journal of Experimental Medicine

Article Title: Macrophages promote anti-androgen resistance in prostate cancer bone disease

doi: 10.1084/jem.20221007

Figure Lengend Snippet: Enzalutamide resistance of bone-metastatic PC can be blocked by SRC-specific inhibitor. (A) Src activity estimated using Src score (see Materials and methods) in RNA-seq data of FACS-purified MycCaP-Bo tumor cells from indicated disease stage/treatment (as described in ). (B) Src score in patient bone metastasis and metastases from other organs. (C) Correlation of Src score with macrophage abundance in patient bone metastasis datasets. (D) Correlation of Src score with ECM score in patient bone metastasis datasets. (E) Correlation of Src score with FN1 expression in patient bone metastasis datasets. (F) Correlation of Src score with ITGA5 expression in patient bone metastasis datasets. (G) Immunoblot showing the level of phosphorylated SRC (pSRC) and total SRC (t-SRC) in MycCaP-Bo cells seeded in wells precoated with 1% BSA (BSA), 1 μg/ml FN1 (FN1-1), and 10 μg/ml FN1 (FN1-10) for indicated time. (H) Immunoblotting showing the level of pSRC and t-SRC in modified MycCaP-Bo cells with doxycycline-induced expression of control shRNA or shRNA-targeting Fn1. The cells were treated with doxycycline (500 ng/ml) for 4 d. (I) Immunoblotting showing the level of pSRC and t-SRC in control MycCaP-Bo cells (Ctrl), MycCaP-Bo cells overexpressing ITGA5 clone 1 (#1) and clone 4 (#4) seeded in wells precoated with 1% BSA (BSA) or 1 μg/ml FN1 (FN1) for 6 h before sample harvest. (J) Immunoblotting showing the level of pSRC and t-SRC in in vivo MycCaP-Bo bone metastasis samples with indicated treatments. (K) Representative BLI images of resistant MycCaP-Bo bone metastasis following eCF506 treatment. (L) BLI quantification of resistant MycCaP-Bo bone metastasis following eCF506 treatment ( n = 8∼10). Data are mean ± SEM; *, P < 0.05; **, P < 0.01; ns, not significant. ANOVA was used in A and B, two-tailed unpaired Student’s t test was used for L, and Pearson correlation analysis was used in C–F. Source data are available for this figure: .

Article Snippet: After blocking in Odyssey Blocking Buffer TBS (927-50000; LI-COR Biosciences), membranes were probed with primary Abs against pSRC (D49G4, #6943; Cell Signaling Technology) or SRC (36D10, #2109; Cell Signaling Technology) overnight at 4°C.

Techniques: Activity Assay, RNA Sequencing Assay, Purification, Expressing, Western Blot, Modification, shRNA, In Vivo, Two Tailed Test

Journal: The EMBO Journal

Article Title: PTEN deficiency exposes a requirement for an ARF GTPase module for integrin‐dependent invasion in ovarian cancer

doi: 10.15252/embj.2023113987

Figure Lengend Snippet: A, B Immunofluorescence and confocal imaging of Trp53 −/− ; Pten −/− 1.15 spheroids stained for α5‐integrin or β1‐integrin (grey or FIRE LUT), Hoechst (blue) and F‐actin (magenta). Magnified images from boxed regions shown. Arrowheads, labelling at protrusion tips. Scale bars, 5 μm. Representative of n = 3 spheroids imaged. (B) Intensity profiles for integrins (grey) and F‐actin (magenta) from spheroids in (A). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead. C, D Immunofluorescence and confocal imaging of Trp53 −/− ; Pten −/− 1.15 spheroids stained for pFAK (Y379) or pSRC Family Kinases (SFK pY416) (grey or FIRE LUT), Hoechst (blue) and F‐actin (magenta). Magnified images from boxed regions shown. Arrowheads, positive staining. Scale bars, 5 μm. Representative of n = 5 spheroids imaged. (D) Intensity profiles for active FAK and Src (grey) and F‐actin (magenta) from spheroids in (C). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead. E–H Representative capture ELISA graphs (E, G) and associated quantitation (F, H) for recycling of internalised cargoes between Trp53 −/− versus Trp53 −/− ; Pten −/− cells or Trp53 −/− ; Pten −/− cells expressing sh Scramble versus sh Arf6 for active β1‐integrin. Graphs shown are representative of n = 2 (E) or n = 3 (G) independent replicates. Data, mean (black square) ± SD for repeated experiments (large circles), 1–3 technical replicates/experiment/timepoint (small circles), two‐tailed t ‐test, P ‐values are annotated. I–K Overall survival (% patients, months; TCGA OV data set) of patients grouped into combined expression based on median mRNA split. (I) Low (red line, M1) or high (blue line, M2) expression for all mRNA, control, remaining patients (green line), (J), same as (I), but CYTH2 Ex9 PSI, rather than total CYTH2. (K), as for (I), but PTEN protein levels split by quantiles (red and blue, Q1 + Q2, Q3, low PTEN, green Q4, high PTEN). Median survival, sample size ( n ) and P ‐value, Log‐rank test (Mantel‐Cox) annotated. L Differential abundance ( x , Log Ratio between conditions; y , Log 10 q ‐values) of proteins in PIP 3 ‐responsive module (ARF6 HI ‐AGAP1 HI ‐CYTH2 2G ) versus PI(4,5)P 2 ‐responsive ARF module (ARF6 HI ‐AGAP1 HI ‐CYTH2 3G ) protein samples. Reverse Phase Protein Array Data, TCGA OV. Significantly altered components in AKT signalling pathway labelled (−Log 10 q ‐value > 1.3). M Schema, molecular model for ARF GTPase regulation of integrin‐dependent invasion. Source data are available online for this figure.

Article Snippet: The following antibodies were added at 1:200 dilution in PFS and incubated overnight at 4°C with gentle shaking: Collagen IV (Abcam, ab19808), pAKT pS473 (CST, 4060, D9E), pFAK pY397 (CST, 3283), pSRC Family pY416 (CST, 2101), V5‐Tag (ABM, G189), PI3Kβ (Proteintech, 21739‐1‐AP), AGAP1 (TFS, 50542), ITGB1 (Merck, MAB1997), ITGA5 (BD Bioscience, 553319).

Techniques: Immunofluorescence, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Expressing, Two Tailed Test, Protein Array

Journal: PLOS ONE

Article Title: Combining MEK and SRC inhibitors for treatment of colorectal cancer demonstrate increased efficacy in vitro but not in vivo

doi: 10.1371/journal.pone.0281063

Figure Lengend Snippet: Western blots performed to detect changes in the levels of the signaling factors pSRC, pFAK, and pERK in multiple KRAS-mutated CRC cell lines are shown. Changes in phosphorylation levels were compared with their respective total levels. Vinculin was used as a loading control. C, control; T, trametinib; D, dasatinib; T+D, trametinib and dasatinib. The numbers below the blots denote the phospho-protein levels relative to total protein levels in each sample (Control cells standardized to 1).

Article Snippet: Antibodies against pERK1/2 (cat. #9101; RRID:AB_331646), ERK1/2 (cat. #4695; RRID:AB_390779), pAKT (cat. #9271; RRID:AB_329825), AKT (cat. #9272; RRID:AB_329827), pSRC (cat. #6943; RRID:AB_10013641), SRC (cat. #2123; RRID:AB_2106047), pFAK (cat. #3284; RRID:AB_10831810), FAK (cat. #3285; RRID:AB_2269034), pMEK (cat. #9154; RRID:AB_2138017), MEK (cat. #9126; RRID:AB_331778), PARP (cat. #9542; RRID:AB_2160739), cleaved PARP (cat. #9541; RRID:AB_331426), caspase 3 (cat. #9665; RRID:AB_2069872), and cleaved caspase 3 (cat. #9661; RRID:AB_2341188) were obtained from Cell Signaling Technology.

Techniques: Western Blot

Journal: bioRxiv

Article Title: A Back-Door Insights into the modulation of Src kinase activity by the polyamine spermidine

doi: 10.1101/2023.01.15.524120

Figure Lengend Snippet: ( A ) Enzymatic activity of rhSrc in the presence of ATP (10 μM), synthetic peptide (100 μM) and increasing concentration of spermidine (45 nM to 100 μM). ADP-Glo™ Kinase Assay (Promega) was used to detect the activity. Results are shown as fold change vs untreated samples (fold change = 1, dotted line). Spermidine EC50=106,4 nM ± 13,4. ( B ) Immunoblot analysis of phosphorylated (pSrc) and total Src protein level evaluated in cell lysates from SYF cells reconstituted with vector coding for wild-type Src and then treated with increasing concentration of spermidine (130 nM to100 μM). Actin expression was used as normalizer. One representative immunoblot of three is shown. ( C ) pSrc/Src ratio of scanning densitometry analysis of three independent immunoblots. Data (mean ± SD) are reported as fold change of samples treated with spermidine relative to untreated cells (fold change = 1, dotted line). Spermidine EC50=6,4 μM ± 0,6. ( D ) Enzymatic activity of rhSrc in the presence of spermidine, with or without ATP and peptide substrate. ( E ) Enzymatic activity of rhSrc in the presence of fixed concentrations of spermidine and increasing concentration of peptide substrate. Data were analyzed with one-way ANOVA followed by post-hoc Bonferroni test. *p < 0.05, ***p < 0.001.

Article Snippet: The pSrc/Src ratio was assessed with a rabbit Phospho-Src Family (Tyr416) Antibody (#2101, Cell Signaling Technology, Danvers, MA, USA; RRID:AB_331697), recognizing the phosphorylation at tyrosine 424 in murine Src, followed by the detection of total Src by rabbit monoclonal antibody (36D10, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_2106059), as previously shown ( ).

Techniques: Activity Assay, Concentration Assay, Kinase Assay, Western Blot, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: A Back-Door Insights into the modulation of Src kinase activity by the polyamine spermidine

doi: 10.1101/2023.01.15.524120

Figure Lengend Snippet: ( A ) Schematic representation of the Src domains and kinase activation. The catalytic activation of the enzyme is characterized by the phosphorylation of the Y424 (pY424) in the activation loop. Created with BioRender.com . ( B ) Electrostatic potential surface of the Src SH2 domain showing the pY binding site (R182, H209) and the putative allosteric site for the endogenous polyamine as delimited by the glutamate residues (E155 and E174). ( C ) Overlay of docking solutions of spermidine into the shallow cavity of Src kinase (poses #1-18, Appendix Table S1). E155 and E174 residues are shown with magenta carbon-atoms. Induced-fit conformations of side chains of residues shaping the cavity are shown with grey carbon-atoms according to each docking solution. Conformations of spermidine according to each docking solution are shown with green carbon-atoms. The Src SH2 domain is shown with magenta cartoon depicting the secondary structure. ( D ) Best energy-scored solution of the binding mode of spermidine into the allosteric pocket of Src (pose #1, Appendix Table S1). E155 and E174 are shown with magenta carbon-atoms. Interacting residues and spermidine are shown with grey and green carbon-atoms, respectively. Hydrogen bond interactions are shown with yellow dashed lines, while the π-cation interaction is reported with green dashed line. ( E ) Immunoblot analysis of phosphorylated (pSrc) and total Src protein level in cell lysates from SYF cells either reconstituted with vector coding for wild-type Src (WT) or Src mutated at glutamate 155 or 174 with alanine (E155A; E174A). Cells were then exposed to increasing concentration of spermidine (130 nM to 100 μM). Actin expression was used as normalizer. One representative immunoblot of three is shown. ( F ) Activation of Src kinase in SYF cells treated as in ( E ) and measured as pSrc/Src ratio of scanning densitometry analysis of three independent immunoblots. Results (mean ± SD) are reported as fold change of samples treated with spermidine relative to untreated cells (fold change = 1, dotted line). ( G ) Schematic representation of the reporter functions. In the presence of active Src kinase, the phosphorylation of Src peptide results in its intra-molecular interaction with the SH2 domain that prevents the complementation of split luciferase fragments and generates a reduced bioluminescence activity. In the absence of Src activation, the N- and C-terminal luciferase domains are reconstituted and thus the bioluminescent activity is restored. ( H ) Measurement of luminescent signal in SYF cells co-expressing the reporter and the wild-type Src or its mutants (E155A and E174A), and then exposed to spermidine (at 10 µM and 100 µM). Results are reported as fold change of bioluminescent signal in stimulated cells as compared to untreated samples. Data ( F, H ) were analyzed with 2-way ANOVA followed by post-hoc Bonferroni test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The pSrc/Src ratio was assessed with a rabbit Phospho-Src Family (Tyr416) Antibody (#2101, Cell Signaling Technology, Danvers, MA, USA; RRID:AB_331697), recognizing the phosphorylation at tyrosine 424 in murine Src, followed by the detection of total Src by rabbit monoclonal antibody (36D10, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_2106059), as previously shown ( ).

Techniques: Activation Assay, Binding Assay, Western Blot, Plasmid Preparation, Concentration Assay, Expressing, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: A Back-Door Insights into the modulation of Src kinase activity by the polyamine spermidine

doi: 10.1101/2023.01.15.524120

Figure Lengend Snippet: ( A ) Immunoblot analysis of total Src protein level in cell lysates from SYF cells either reconstituted with vector coding for wild-type Src (WT) or Src mutated at glutamate 155 or 174 with alanine (E155A; E174A). SYF cells transfected with empty vector (SYF) were used as control. Actin expression was used as normalizer. ( B ) Immunoblot analysis of phosphorylated (pSrc) and total Src protein level in cell lysates from SYF cells either reconstituted with vector coding for wild-type Src (WT) or Src mutated at glutamate 155 or 174 with alanine (E155A; E174A). Cells were then exposed to LPA (20 μM) for the indicated time. β-tubulin expression was used as normalizer.

Article Snippet: The pSrc/Src ratio was assessed with a rabbit Phospho-Src Family (Tyr416) Antibody (#2101, Cell Signaling Technology, Danvers, MA, USA; RRID:AB_331697), recognizing the phosphorylation at tyrosine 424 in murine Src, followed by the detection of total Src by rabbit monoclonal antibody (36D10, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_2106059), as previously shown ( ).

Techniques: Western Blot, Plasmid Preparation, Transfection, Expressing